CN104034782A - Potential sensor for detecting dichlorotris (1,10-phenanthroline) ruthenium (II) and detection method and application of potential sensor - Google Patents
Potential sensor for detecting dichlorotris (1,10-phenanthroline) ruthenium (II) and detection method and application of potential sensor Download PDFInfo
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Abstract
The invention relates to a sensor, and particularly relates to a potential sensor for detecting dichlorotris (1,10-phenanthroline) ruthenium (II) and a detection method and application of the potential sensor. The potential sensor comprises a working electrode, a reference electrode and a potential determinator, wherein the potential determinator is respectively connected with the working electrode and the outer reference electrode through lead wires; the working electrode and the outer reference electrode are inserted into a detection pool containing a detection solution, a polymer sensitive membrane is adhered to the bottom of the working electrode, and a power device which plays a role in solution disturbance is arranged in the detection pool. The potential sensor for detecting the dichlorotris (1,10-phenanthroline) ruthenium (II), which is disclosed by the invention, is high in sensitivity and high in selectivity and can be used for biological sensing based on DNA. According to the potential sensor, different target molecules can be detected by selecting different nucleic acid aptamers by the biological sensing based on DNA, when in detection, the sensor can be combined with circulating isothermal chain displacement polymerization reaction, hybrid chain reaction and a nanometer material amplification technology, so that the detection sensitivity and the detection selectivity are further enhanced.
Description
Technical field
The present invention relates to sensor, a kind of detection dichloro three (1 specifically, 10-phenanthrolene) ruthenium (II) (potentiometric sensor and detection method and the application of Dichlorotris (1,10-phenanthroline) ruthenium (II)).
Background technology
Dichloro three (1,10-phenanthrolene) ruthenium (II) is a kind of efficient chemical illuminating reagent, and widespread use is based on chemiluminescent analytical technology.Yet above-mentioned chemiluminescence is easily subject to the color of sample and the impact of turbidity, thereby limited its widespread use.Potentiometric detection sensitivity based on polymer sensitive membrane ion-selective electrode is high, selectivity good, fast response time, and testing process is not disturbed by the color of sample and turbidity.Thereby develop high-sensitive dichloro three (1,10-phenanthrolene) ruthenium (II) polymer sensitive membrane ion-selective electrode and there is important using value.
That polymer film ion selective electrode has is easy and simple to handle, response fast, the advantage such as equipment is inexpensive, be particularly suitable for the detection of Site Detection and gross sample, be widely used in the direct mensuration of various electrolyte ions in whole blood, serum, urine, tissue, intracellular fluid and dilution thereof.Yet its application focuses mostly on and detects at electrolyte ion.Aptamer (Aptamer) refers to that the part evolution technology of utilization index enrichment filters out target is had to the interactional oligonucleotides of specificity (DNA or RNA) from specific oligonucleotide library, as a kind of novel molecular recognition elements, can be efficiently, specifically in conjunction with various target molecules, have easily synthetic, easily store and the advantage such as good stability; Yet aptamer itself does not have the function of display.Detection method based on aptamer needs mark fluorescent, nano material conventionally at present, thereby can produce detection signal, and this class detection method one side testing cost is high, complex operation; Labeling process has also affected the binding ability of aptamer on the other hand.
Summary of the invention
The object of the invention is to provide a kind of detection dichloro three (1,10-phenanthrolene) ruthenium (II) (potentiometric sensor and detection method and the application of Dichlorotris (1,10-phenanthroline) ruthenium (II)).
For achieving the above object, the technical solution used in the present invention is:
A kind of detection dichloro three (1,10-phenanthrolene) potentiometric sensor of ruthenium (II), comprise working electrode, contrast electrode and potential measurement instrument, it is characterized in that: potential measurement instrument connects respectively working electrode and outer contrast electrode by wire, working electrode and outer contrast electrode insert and fill in the detection cell that detects liquid, working electrode bottom is provided with polymer sensitive membrane, is provided with the propulsion system with disturbance solution effects in detection cell;
Described polymer sensitive membrane is mixed by the ratio of weight and number of 20-40:40-80:0.1-10:0.1-10 by polymeric matrix material, plastifier and cationite, then be dissolved in tetrahydrofuran solution, after being mixed, at room temperature place 12-24h, obtain resilient polymer sensitive membrane;
Described polymeric matrix material is Polyvinylchloride, carboxylated Polyvinylchloride, poly-butylacrylic acid ester, butyl polyacrylate, polyetherimide, rubber, sol-gel film, methyl methacrylate-certain herbaceous plants with big flowers ylmethyl methyl acrylate copolymer or n-butyl acrylate-hydroxyethyl methylacrylate multipolymer; Plastifier is o-nitrobenzene octyl ether, two-2-ethylhexyl ester in the last of the ten Heavenly stems, dibutyl sebacate or di-n-octyl sebacate; Cationite is four (3,5-bis-(trifluoromethyl) phenyl) sodium borate, dinonylnaphthalene sulfonic acid or dinonylnaphthalene sulfonic acid salt or borate.
Between described working electrode and polymer sensitive membrane, be provided with conducting stratum material.
Described working electrode is liquid electrode or solid electrode; Wherein, liquid working electrode fills internal-filling liquid and is inserted with the electrode of internal reference electrode in being; When working electrode is liquid electrode, sensitive membrane is dissolved in tetrahydrofuran solution, at room temperature places 12-24h after being mixed, and then attaches to electrode holder bottom; When working electrode is solid electrode, sensitive membrane is dissolved in methylene chloride (logical nitrogen deoxygenation in advance), and membrane material drips and is coated in solid electrode lower surface.
Described solid working electrode is carbon electrode, gold electrode, copper electrode, silver electrode, platinum electrode, indium-tin oxide electrode, zinc oxide electrode, silicon electrode, carbon fiber electrode, silicon carbide electrode, porous gold electrode, carbon nano tube modified electrode, gold nano modified electrode, platinum Nanoparticle Modified Electrode, graphene modified electrode, C60 modified electrode, porous carbon modified electrode, carbon screen printing electrode, golden screen printing electrode, refill plate electrode or ceramic chip electrode;
Described conducting stratum material is 3-alkylthrophene, polypyrrole, polythiophene, polyaniline, polyacetylene, poly-n-octyl thiophene, six polythiophenes, poly-ethylenedioxy thiophene, carbon nano-tube, Graphene, platinum nanometer, gold nano, molybdenum disulfide, mesoporous carbon, porous gold or tin indium oxide nanometer.
A kind of detection dichloro three (1,10-phenanthrolene) method of ruthenium (II), the working electrode of described potentiometric sensor and the insertion of outer contrast electrode are contained to dichloro three (1 to be measured, 10-phenanthrolene) in the detection liquid of ruthenium (II), by potential measurement instrument, measure the potential change on polymer sensitive membrane on working electrode, according to potential change signal and standard working curve, record dichloro three to be measured (1,10-phenanthrolene) ruthenium (II) concentration.
The application of the potentiometric sensor of a kind of detection dichloro three (1,10-phenanthrolene) ruthenium (II), described potentiometric sensor can be used as the biology sensor based on DNA or aptamer, the detection quantitative to different target molecules.
Be further, using aptamer as identification molecule, dichloro three (1,10-phenanthrolene) ruthenium (II) is as embedding molecule, aptamer and complementary DNA form double-stranded DNA, dichloro three (1,10-phenanthrolene) ruthenium (II) embeds double-stranded DNA, by the potentiometer in described potentiometric sensor, measure the indication ion dichloro three (1 causing after target molecule and aptamer effect, 10-phenanthrolene) potential change that ruthenium (II) concentration change causes, records target molecule testing concentration according to potential change signal and standard working curve.
Further, add dichloro three (1,10-phenanthrolene) ruthenium (II) concentration 10
-1-10
-9mole, with double-stranded DNA interface interaction time 0.1-10 hour, measure the potential response that remains dichloro three (1,10-phenanthrolene) ruthenium (II) generation in solution; Then measure after target molecule and aptamer effect, add dichloro three (1,10-phenanthrolene) ruthenium (II) concentration 10
-1-10
-9mole, with double-stranded DNA interface interaction time 0.1-10 hour, in solution, remain the potential response that dichloro three (1,10-phenanthrolene) ruthenium (II) produces, the electric potential signal of both differences records the concentration of target molecule by standard working curve according to potential change signal.
Or, add dichloro three (1,10-phenanthrolene) ruthenium (II) concentration 10
-1-10
-9mole, with double-stranded DNA interface interaction time 0.1-10 hour, then by adjusting the temperature to 95 degree, interface double-stranded DNA unwinds, discharge dichloro three (1,10-phenanthrolene) ruthenium (II), to buffer solution, is measured the potential response that in buffer solution, dichloro three (1,10-phenanthrolene) ruthenium (II) produces; Measure after target molecule and aptamer effect, add dichloro three (1,10-phenanthrolene) ruthenium (II) concentration 10
-1-10
-9mole, with double-stranded DNA interface interaction time 0.1-10 hour, then by adjusting the temperature to 95 degree, interface double-stranded DNA unwinds, discharge dichloro three (1,10-phenanthrolene) ruthenium (II) to buffer solution, measure the dichloro three (1 that buffer solution double center chain DNA discharges, 10-phenanthrolene) potential response that ruthenium (II) produces, both differences record the concentration of target molecule by standard working curve according to potential change signal.
Described aptamer is incorporated in liquid to be detected, or is incorporated in and detects in liquid after fixing with substrate.
Described fixed nucleic acid is fit and the substrate of complementary DNA is ferroferric oxide magnetic nanoparticle, at the bottom of magnetic graphite thiazolinyl, carbon substrate, gold substrate, copper substrate, at the bottom of money base, platinum substrate, tin indium oxide substrate, at the bottom of Zinc oxide-base, carbon fiber substrates, silicon carbide substrate, porous gold substrate, carbon nano tube modified substrate, gold nano is modified substrate, the nano-modified substrate of platinum, graphene modified substrate, C60 modifies substrate, porous carbon is modified the surfaces such as substrate, carbon filament reticulated printing electrode basement, spun gold reticulated printing electrode basement, the substrate of refill plate electrode or ceramic chip electrode basement.
It is that aptamer and wall scroll complementary strand form double-stranded DNA that described aptamer and complementary DNA form double-stranded DNA; Or aptamer forms long double-stranded DNA by circulation isothermal strand displacement polyreaction and hybridization chain reaction (Hybridization chain reaction, HCR).
Described aptamer comprises: take all kinds of aptamers that heavy metal ion, organic dyestuff, amino acid, organic molecule, nucleic acid, RNA, polysaccharide, enzyme, growth factor, antibody, bacterium, cell, virus be part.Wherein, aptamer can also replace with the DNA that contains specific base sequence.
The present invention has advantages of:
Provided by the invention for detection of dichloro three (1,10-phenanthrolene) (Dichlorotris (1 for ruthenium (II), 10-phenanthroline) ruthenium (II)) potentiometric sensor is highly sensitive, selectivity is high, and this potentiometric sensor can be used in the bio-sensing based on DNA simultaneously; Bio-sensing based on DNA can be by selecting different aptamers, can realize the detection of different target molecules, during detection, can combine with circulation isothermal strand displacement polyreaction and hybridization chain reaction and nano material amplifying technique, further improve sensitivity, the selectivity detecting.
Separately, dichloro three (1,10-phenanthrolene) ruthenium (II) is a kind of little molecule, can optionally embed double-stranded DNA.Aptamer is a kind ofly can identify efficiently, specifically the DNA fragmentation of certain target molecules.DNA single chain by introducing with aptamer complementation, forms DNA double chain, and now dichloro three (1,10-phenanthrolene) ruthenium (II) can optionally embed double-stranded DNA.Before and after target molecule and aptamer effect, changed in solution or the concentration of aptamer functionalization substrate surface dichloro three (1,10-phenanthrolene) rutheniums (II), thereby the variation that produces electrode potential realize the detection of target molecule accordingly.By selecting different aptamers, the method has certain versatility, can realize the detection of different target molecules.By introducing circulation isothermal strand displacement polyreaction and hybridization chain reaction (Hybridization chain reaction, HCR), form the double-stranded DNA of length, or a large amount of DNA of nano material load, realize the amplification of potentiometric detection signal.
Accompanying drawing explanation
The sensor schematic diagram that Fig. 1 provides for the embodiment of the present invention.
The sensor schematic diagram that Fig. 2 provides for the embodiment of the present invention.
The liquid membrane electrode that Fig. 3 provides for the embodiment of the present invention detects dichloro three (1,10-phenanthrolene) ruthenium (II) potential response figure in real time.
The solid contact electrode that Fig. 4 provides for the embodiment of the present invention detects dichloro three (1,10-phenanthrolene) ruthenium (II) potential response figure in real time.
The magnetic Nano surface aptamer that Fig. 5 provides for the embodiment of the present invention and complementary DNA hybridization chain reaction form long double-stranded DNA figure.
Embodiment
Embodiment 1
Potentiometric sensor, comprises working electrode 1, outer contrast electrode 2, internal reference electrode 3 and potential measurement instrument 4, and potential measurement instrument 4 connects respectively internal reference electrode 3 and outer contrast electrode 2 by wire, and internal reference electrode 3 insertions fill internal-filling liquid 8, and (internal-filling liquid is 10
-3mole dichloro three (1,10-phenanthrolene) in working electrode 1 ruthenium (II)), working electrode 1 and outer contrast electrode 2 insert and fill in the detection cell 5 that detects liquid 6, working electrode 1 is for being fixed with the electrode holder of polymer sensitive membrane 7, in detection cell 5, be provided with magnetic stirring 9 with beating action, supporting at the bottom of detection cell have magnetic stirring apparatus to rotate magnetic stir bar.The electrode holder that is fixed with polymer sensitive membrane 7 is polyvinyl chloride pipe.
Detection method:
A. with cationite four (3,5-bis-(trifluoromethyl) phenyl) polymer film of sodium borate doping is working electrode sensitive membrane, silver-silver chloride electrode is inside and outside contrast electrode, internal reference electrode is connected with the working electrode joint of electrochemical workstation CHI760D, outer contrast electrode is connected (referring to Fig. 1) with contrast electrode joint, selects " open circuit potential-time " technology to measure potential value.
The preparation of working electrode sensitive membrane: according to mass percent 1%:33%:66% by four (3,5-bis-(trifluoromethyl) phenyl) sodium borate, Polyvinylchloride, o-nitrobenzene octyl ether mix, gained potpourri is 345mg altogether, be dissolved in 3.5mL tetrahydrofuran solution, under room temperature, in flat glass ring (internal diameter is 3.6cm), place, after about 8h, tetrahydrofuran evaporates, and obtains uniform polymer sensitive membrane.
Be fixed with the preparation of the electrode holder working electrode of polymer sensitive membrane: above-mentioned resulting polymers sensitive membrane is beaten to the polymer sensitive membrane of getting diameter 0.7cm with card punch, and by beating the film of getting, be fixed on polyvinyl chloride pipe bottom by tetrahydrofuran.Electrode is used front 10
-3the about 8h of activation in M dichloro three (1,10-phenanthrolene) ruthenium (II).
B. under stirring condition, measure electrode 10
-3-10
-9m standard solution (10
-3-10
-9m standard solution is 10
-3-10
-9the dichloro three (1 of M, 10-phenanthrolene) potential response (referring to Fig. 3) ruthenium (II)), according to potential response, make the standard working curve of potential changing value to dichloro three (1,10-phenanthrolene) ruthenium (II) concentration.
C. electrode is inserted to unknown concentration dichloro three (1,10-phenanthrolene) in the analyte sample fluid of ruthenium (II), the response of recording electrode current potential, then tries to achieve the concentration of dichloro three (1,10-phenanthrolene) ruthenium (II) according to typical curve.
Embodiment 2
Potentiometric sensor, comprise working electrode 1, contrast electrode 2 and potential measurement instrument 3, potential measurement instrument 3 connects respectively working electrode 1 and outer contrast electrode 2 by wire, working electrode 1 and outer contrast electrode 2 insert and fill in the detection cell 4 that detects liquid, working electrode 1 is for being fixed with the solid electrode of polymer sensitive membrane 5, in detection cell 4, be provided with magnetic stirring 6 with beating action, supporting at the bottom of detection cell have magnetic stirring apparatus to rotate magnetic stir bar (referring to Fig. 2).
Detection method:
A. the polymer film with the doping of cationite four (3,5-bis-(trifluoromethyl) phenyl) sodium borate is working electrode sensitive membrane, and silver-silver chloride electrode is outer contrast electrode, selects " open circuit potential-time " technology to measure potential value.
By 50mg (wherein four (3,5-bis-(trifluoromethyl) phenyl) sodium borate 0.5mg, four (dodecyl)-tetra-(4-chlorphenyl) ammonium borates (ETH500) 5mg, polymer film methyl methacrylate-certain herbaceous plants with big flowers ylmethyl methyl acrylate 44.5mg) membrane component is dissolved in 0.5mL methylene chloride (in advance logical nitrogen deoxygenation), stirs after two hours, lead to again after nitrogen deoxygenation standby.Get the above-mentioned membrane material of 100 microlitre and drip on the solid carbon electrode of polishing, and more than this electrode is placed in to thermostatic drying chamber volatile dry 12h.
Dried electrode can be soaked in 10
-4moL/L Ru
2+after activating 48h in solution, measure.During determination of electrode, use Ag-AgCl to make contrast electrode, during mensuration: solid electrode | polymer sensitive membrane || sample solution | 3M KCl solution | Ag-AgCl.
B. under stirring condition, measure electrode 10
-3-10
-10potential response in M standard solution, according to potential response (referring to Fig. 4), makes the standard working curve of potential changing value to dichloro three (1,10-phenanthrolene) ruthenium (II) concentration.
C. electrode is inserted to the testing sample of unknown concentration dichloro three (1,10-phenanthrolene) ruthenium (II), the response of recording electrode current potential, then tries to achieve the concentration of dichloro three (1,10-phenanthrolene) ruthenium (II) according to typical curve.
Embodiment 3
Be with embodiment 1 and 2 differences: the current potential sensing of the present embodiment development based on above-described embodiment, and with aptamer identification and dichloro three (1,10-phenanthrolene) ruthenium (II) is indication ion, adopt dichloro three (1, the 10-phenanthrolene) ruthenium (II) of development as the current potential method for sensing of detecting device.It is example that the present embodiment take that agricultural chemicals buttocks worm amidine detects, and detection method is as follows:
Current potential sensing: comprise working electrode 1, contrast electrode 2 and potential measurement instrument 3, potential measurement instrument 3 connects respectively working electrode 1 and outer contrast electrode 2 by wire, working electrode 1 and outer contrast electrode 2 insert and fill in the detection cell 4 that detects liquid, working electrode 1 is for being fixed with the solid electrode of polymer sensitive membrane 5, in detection cell 4, be provided with magnetic stirring 6 with beating action, supporting at the bottom of detection cell have magnetic stirring apparatus to rotate magnetic stir bar (referring to Fig. 2).
Polymer film with the doping of cationite four (3,5-bis-(trifluoromethyl) phenyl) sodium borate is working electrode sensitive membrane, and silver-silver chloride electrode is outer contrast electrode, selects " open circuit potential-time " technology to measure potential value.
10 μ L25mmol/LPOT-CH2Cl2 solution are dripped in gold electrode surfaces, after bone dry again by 50mg (wherein four (3,5-bis-(trifluoromethyl) phenyl) sodium borate 0.5mg, four (dodecyl)-tetra-(4-chlorphenyl) ammonium borates (ETH500) 5mg, polymer film methyl methacrylate-certain herbaceous plants with big flowers ylmethyl methyl acrylate 44.5mg) membrane component is dissolved in 0.5mL methylene chloride (in advance logical nitrogen deoxygenation), stirs after two hours, lead to again after nitrogen deoxygenation standby.Get the above-mentioned membrane material of 100 microlitre and drip on the solid gold electrode of polishing, and more than this electrode is placed in to thermostatic drying chamber volatile dry 12h.Dried electrode can be soaked in 10
-4moL/LRu
2+after activating 48h in solution, measure.
Detect:
(a) preparation at aptamer functionalization double-stranded DNA interface: working electrode is placed in to 10
-6m5' holds in mercapto-functionalized aptamer (the buttocks worm amidine of take is example) solution, and aptamer, electrode surface reaction 24 hours, is further placed in 10 by gold electrode
-6in the sulfydryl hexanol of M, react 12h, above-mentioned gold electrode is placed in to 10
-6the DNA effect 2h that M and buttocks worm amidine aptamer part are complementary, obtains aptamer functionalization double-stranded DNA interface.
(b) Criterion working curve: step a gained double-stranded DNA interface is placed in 1mL10
-5in mole dichloro three (1,10-phenanthrolene) ruthenium (II) solution, act on 1 hour, interface is separated with solution, and working electrode is placed in to separated rear solution, measures in solution and remains dichloro three (1,10-phenanthrolene) potential response that ruthenium (II) produces, as blank.
Step a gained double-stranded DNA interface and 10
-3-10
-10after mole Acetamiprid standard solution effect 1h, interface is separated with solution, is placed in 1mL10 with the reacted double-stranded DNA of above-mentioned standard solution interface
-5mole dichloro three (1,10-phenanthrolene) in ruthenium (II) solution, act on 1 hour, interface is separated with solution, working electrode is placed in to separated rear solution, measure in solution and remain the potential response that dichloro three (1,10-phenanthrolene) ruthenium (II) produces, as the potential response of standard solution.
According to remaining potential response that dichloro three (1,10-phenanthrolene) ruthenium (II) produces in standard solution in the potential change signal Criterion working curve of blank difference.
(c) mensuration of the testing sample of unknown concentration Acetamiprid:
Step a gained double-stranded DNA interface is placed in 1mL10
-5in mole dichloro three (1,10-phenanthrolene) ruthenium (II) solution, act on 1 hour, interface is separated with solution, and working electrode is placed in to separated rear solution, measures in solution and remains dichloro three (1,10-phenanthrolene) potential response that ruthenium (II) produces, as blank.
After the testing sample effect 1h of step a gained double-stranded DNA interface and unknown concentration Acetamiprid, interface is separated with solution, is placed in 1mL10 with the reacted double-stranded DNA of above-mentioned testing sample interface
-5mole dichloro three (1,10-phenanthrolene) in ruthenium (II) solution, act on 1 hour, interface is separated with solution, working electrode is placed in to separated rear solution, measure in solution and remain the potential response that dichloro three (1,10-phenanthrolene) ruthenium (II) produces, as the potential response of testing sample.
In testing sample solution, remain the potential response of dichloro three (1,10-phenanthrolene) ruthenium (II) generation in blank difference, difference, again by standard working curve, finally obtains recording the concentration of Acetamiprid in testing sample.
Or,
Step a gained double-stranded DNA interface is placed in 1mL10
-5in mole dichloro three (1,10-phenanthrolene) ruthenium (II) solution, act on 1 hour, interface is separated with solution, then again interface is placed in to pH7.410
-3in mole of phosphoric acid salt buffer solution, be heated to 95 degree, the dichloro three (1 of combination on interface, 10-phenanthrolene) ruthenium (II) is discharged in buffer solution, again working electrode is placed in to buffer solution, measure dichloro three (1 in buffer solution, 10-phenanthrolene) potential response that ruthenium (II) produces, as blank.
After the testing sample effect 1h of step a gained double-stranded DNA interface and unknown concentration Acetamiprid, interface is separated with solution, is placed in 1mL10 with the reacted double-stranded DNA of above-mentioned testing sample interface
-5in mole dichloro three (1,10-phenanthrolene) ruthenium (II) solution, act on 1 hour, interface is separated with solution, then again interface is placed in to pH7.410
-3in mole of phosphoric acid salt buffer solution, be heated to 95 degree, the dichloro three (1 of combination on interface, 10-phenanthrolene) ruthenium (II) is discharged in buffer solution, again working electrode is placed in to buffer solution, measure dichloro three (1 in buffer solution, 10-phenanthrolene) potential response that ruthenium (II) produces, in testing sample solution, be discharged into the dichloro three (1 of buffer solution, 10-phenanthrolene) potential response that ruthenium (II) produces is in blank difference, difference, again by standard working curve, finally obtains recording the concentration of Acetamiprid in testing sample.
Aptamer sequence 5-(SH)-(CH of Acetamiprid
2)
6the complementary DNA sequence dna of-TGTAA TTTGTCTGCAG CGGTTCTTGATCGCTGA-CACCATATTATGAA GA-3. and buttocks worm amidine aptamer part is: 3-AAGAACTAGC GACTGTGGR ATAATAC TTCT-5
Embodiment amplifying nucleic acid is fit also can be by heavy metal ion as potassium, mercury, lead, copper, zinc, uranium; Organic dyestuff is as sulfo group rhodamine B, reactive dark green KE-4BD; Amino acid is as arginine, TYR amine; Organic molecule is as ***e, cholic acid, Aspartame (containing phenylalanine), (R)-Thalidomide, 17 beta estradiols, bisphenol-A, buttocks worm amidine; Nucleic acid is as atriphos, adenylate, adenosine diphosphate (ADP); RNA is as TAR-RNA; Polysaccharide is as cellobiose, saliva lactose; Enzyme is as fibrin ferment, protein kinase, centriole cell elastase, HIV1-RT; Toxin is as ricin (WA); Growth factor is as basic fibroblast growth factor, platelet-derived growth factor-B chain; Antibody is as human immunoglobulin(HIg) IgE; All kinds of aptamers that bacterium and cell are part as spore, lung carcinoma cell, pathogenic bacteria are replaced.
Embodiment 4
Adopt the working electrode of embodiment 3, with aptamer, combine with DNA hybridization chain reaction, realize potentiometric detection signal and amplify.The present embodiment be take bisphenol-A detection as example,
Detection method is as follows:
A. the magnetic Nano of 50ul isosulfocyanate radical being modified is placed in 5 * 10
-6in aptamer (take bisphenol-A as the example) solution of M5' end amino functional, 37 degree reactions 1 hour, magnetic resolution, the solution that more above-mentioned aptamer modified magnetic Nano is placed in to 5% bovine serum albumin(BSA) reacts 1 hour, and magnetic resolution obtains aptamer functional magnetic nano particle.Aptamer functional magnetic nano particle is reacted 2 hours with auxiliary DNA solution, on magnetic Nano surface, there is hybridization chain reaction, magnetic resolution, is placed in DNA hybridization buffer solution (10mM Tris-HCl+1mM EDTA+500mM NaCl+1mM MgCl by magnetic Nano
2, pH7.4) in, built aptamer functionalization double-stranded DNA interface.
Wherein, auxiliary DAN solution, for assisting DNA2 and DNA3 reaction after 2 hours, adds auxiliary DNA1 to obtain.
(b) Criterion working curve: after 30 μ L step a gained double-stranded DNA interfacial separation, be placed in 1mL10
-5in mole dichloro three (1,10-phenanthrolene) ruthenium (II) solution, act on 1 hour, interface is separated with solution, and working electrode is placed in to separated rear solution, measures in solution and remains dichloro three (1,10-phenanthrolene) potential response that ruthenium (II) produces, as blank.
Step a gained double-stranded DNA interface and 10
-3-10
-12after M bisphenol-A standard solution effect 1h, interface is separated with solution, is placed in 1mL10 with the reacted double-stranded DNA of above-mentioned standard solution interface
-5mole dichloro three (1,10-phenanthrolene) in ruthenium (II) solution, act on 1 hour, interface is separated with solution, working electrode is placed in to separated rear solution, measure in solution and remain the potential response that dichloro three (1,10-phenanthrolene) ruthenium (II) produces, as the potential response of standard solution.
According to remaining potential response that dichloro three (1,10-phenanthrolene) ruthenium (II) produces in standard solution in the potential change signal Criterion working curve of blank difference.
(c) mensuration of the testing sample of unknown concentration bisphenol-A:
After 30 μ L step a gained double-stranded DNA INTERFACE MAGNETISM separation, be placed in 1mL10
-5in mole dichloro three (1,10-phenanthrolene) ruthenium (II) solution, act on 1 hour, interface is separated with solution, and working electrode is placed in to separated rear solution, measures in solution and remains dichloro three (1,10-phenanthrolene) potential response that ruthenium (II) produces, as blank.
After the testing sample effect 1h of step a gained double-stranded DNA interface and unknown concentration bisphenol-A, interface is separated with solution, is placed in 1mL10 with the reacted double-stranded DNA of above-mentioned testing sample interface
-5mole dichloro three (1,10-phenanthrolene) in ruthenium (II) solution, act on 1 hour, interface is separated with solution, working electrode is placed in to separated rear solution, measure in solution and remain the potential response that dichloro three (1,10-phenanthrolene) ruthenium (II) produces, as the potential response of testing sample
In testing sample solution, remain the potential response of dichloro three (1,10-phenanthrolene) ruthenium (II) generation in blank difference, difference, again by standard working curve, finally obtains recording the concentration of bisphenol-A in testing sample.
In described step a, HCR reaction following (as shown in Figure 5):
(1) isosulfocyanate radical end magnetic particle solution is purchased from Xi'an gold magnetic nano biological science and technology limited Company (xMag
tM).In experiment, according to kit standard program, carry out aptamer mark.Get 2 1.5mL centrifuge tubes and be once labeled as 1,2, then add respectively the good xMag of 30 μ L mark
tMisosulfocyanate radical end magnetic particle solution, magnetic resolution is removed supernatant.
(2) in centrifuge tube 1, add 30 microlitre DNA hybridization buffer solution (10mM Tris-HCl+1mM EDTA+500mM NaCl+1mM MgCl
2, pH7.4), in centrifuge tube 2, add 30 microlitre DNA1 liquid, in 37 ℃ of water-baths, react 1h, then magnetic resolution is got supernatant.
(3), for the centrifuge tube in step 2, add 40 microlitre DNA hybridization buffer solution (10mM Tris-HCl+1mM EDTA+500mM NaCl+1mM MgCl in 1
2, pH7.4), add 40 microlitre DNA2 and DNA3 hybridization reaction solution (adding front hybridization reaction time 2h) in 2, in 37 ℃ of water-baths, react 2h, then magnetic resolution is removed supernatant.Wherein bisphenol-A aptamer sequence is as follows: 5'-GGG CCG TTC GAA CAC GAG CAT GCC GGT GGG TGG TCA GGT GGG ATA GCG TTC CGC GTA TGG CCC AGC GCA TCA CGG GTT CGC ACC AGG ACA GTA CTC AGG TCA TCC TAG-3', the sequence of auxiliary DNA1: 3'-GCG TGG TCC TGT CAT GAG TCC AGT AGG ATC ACT AAA AGG GTC TGA GGG-5'; The sequence of auxiliary DNA2: 5'-TGA TTT TCC CAG ACT CCC CGT GGA CCC CCT CAT-3'; The sequence of auxiliary DNA3: 3'GCA CCT GGG GGA GTA ACT AA A AGG GTC TGA GGG-5'.
Embodiment amplifying nucleic acid is fit also can be by heavy metal ion as potassium, mercury, lead, copper, zinc, uranium; Organic dyestuff is as sulfo group rhodamine B, reactive dark green KE-4BD; Amino acid is as arginine, TYR amine; Organic molecule is as ***e, cholic acid, Aspartame (containing phenylalanine), (R)-Thalidomide, 17 beta estradiols, bisphenol-A, buttocks worm amidine; Nucleic acid is as atriphos, adenylate, adenosine diphosphate (ADP); RNA is as TAR-RNA; Polysaccharide is as cellobiose, saliva lactose; Enzyme is as fibrin ferment, protein kinase, centriole cell elastase, HIV1-RT; Toxin is as ricin (WA); Growth factor is as basic fibroblast growth factor, platelet-derived growth factor-B chain; Antibody is as human immunoglobulin(HIg) IgE; All kinds of aptamers that bacterium and cell are part as spore, lung carcinoma cell, pathogenic bacteria are replaced.
Embodiment amplifying nucleic acid is fit also can be fixed on carbon nano-tube, gold nano, ferroferric oxide magnetic nanoparticle, graphene-supported magnetic nanoparticle, Graphene surface, gold electrode surfaces or carbon electrodes.
Embodiment 5
Adopt the working electrode of embodiment 3, take that nuclease and radical damage DNA detect is example, and detection method is as follows:
A. the preparation at aptamer functionalization double-stranded DNA interface: working electrode is placed in to 10
-6m5' holds in mercapto-functionalized random series DNA solution, and DNA, electrode surface reaction 24 hours, is further placed in 10 by gold electrode
-6in the sulfydryl hexanol of M, react 12h, above-mentioned gold electrode is placed in to 10
-6m and 5' hold the mercapto-functionalized complementary DNA effect 2h of above-mentioned random series DNA part, obtain DNA functionalization double-stranded DNA interface.
B. Criterion working curve: step a gained double-stranded DNA interface is placed in 1mL10
-5in mole dichloro three (1,10-phenanthrolene) ruthenium (II) solution, act on 1 hour, interface is separated with solution, and working electrode is placed in to separated rear solution, measures in solution and remains dichloro three (1,10-phenanthrolene) potential response that ruthenium (II) produces, as blank.
Step a gained double-stranded DNA interface is respectively at the nuclease effect 10min of 0.1U/mL, 1U/mL, 5U/mL, 10U/mL, 30U/mL, 50U/mL, then add respectively 1mM ethylenediamine tetraacetic acid (EDTA) to stop enzyme reaction, interface is separated with solution, is placed in 1mL10 with the double-stranded DNA interface after the nuclease solution reaction of above-mentioned variable concentrations
-5mole dichloro three (1,10-phenanthrolene) in ruthenium (II) solution, act on 1 hour, interface is separated with solution, working electrode is placed in to separated rear solution, measure in solution and remain the potential response that dichloro three (1,10-phenanthrolene) ruthenium (II) produces, as the potential response of standard solution.
According to remaining potential response that dichloro three (1,10-phenanthrolene) ruthenium (II) produces in standard solution in the potential change signal Criterion working curve of blank difference.
(c) mensuration of the testing sample of unknown concentration sample enzyme: step a gained double-stranded DNA interface is placed in 1mL10
-5in mole dichloro three (1,10-phenanthrolene) ruthenium (II) solution, act on 1 hour, interface is separated with solution, and working electrode is placed in to separated rear solution, measures in solution and remains dichloro three (1,10-phenanthrolene) potential response that ruthenium (II) produces, as blank.
After step a gained double-stranded DNA interface and sample enzyme solutions effect 10min to be measured, add 1mM ethylenediamine tetraacetic acid (EDTA) to stop enzyme reaction, interface is separated with solution, is placed in 1mL10 with the reacted double-stranded DNA of above-mentioned testing sample interface
-5mole dichloro three (1,10-phenanthrolene) in ruthenium (II) solution, act on 1 hour, interface is separated with solution, working electrode is placed in to separated rear solution, measure in solution and remain the potential response that dichloro three (1,10-phenanthrolene) ruthenium (II) produces, as the potential response of testing sample.
In testing sample solution, remain the potential response of dichloro three (1,10-phenanthrolene) ruthenium (II) generation in blank difference, difference by standard working curve, finally obtains recording sample enzyme activity in testing sample again.
In step a, the sequence of aptamer/DNA can design according to the selectively acting site of enzyme.As contained 5'-TTA in the base sequence of restriction enzyme A nal ... TAA3', clipped is random series, contains 3'-AAT with it in complementary sequence ... ATT5'.Other restriction enzyme designs according to mentioned above principle.
In step b, determinand can also for the interactional material of aptamer/DNA, the potpourri that comprises deoxyribonuclease, restriction enzyme, exonuclease, ligase, s1 nuclease, nuclease and its inhibitor, hydroxyl radical free radical, or antioxidant and hydroxyl radical free radical mixed solution.
Embodiment 6
Adopt the working electrode of embodiment 3, take atriphos detection as example, adopt Graphene as the embedding carrier of dichloro three (1,10-phenanthrolene) ruthenium (II), detection method is as follows:
(a) preparation at aptamer functionalization DNA interface: working electrode is placed in to 10
-6in the aptamer (take adenosine triphosphate atp as example) of M5' end amino or carboxyl-functional, aptamer, electrode surface reaction 24 hours, is further placed in 10 by gold electrode
-6in the sulfydryl hexanol of M, react 12h,
(b) Criterion working curve: step a gained DNA interface is placed in 1mg/mL graphene solution 2h, is then placed in 1mL10 again
-5in mole dichloro three (1,10-phenanthrolene) ruthenium (II) solution, act on 1 hour, interface is separated with solution, and working electrode is placed in to separated rear solution, measures in solution and remains dichloro three (1,10-phenanthrolene) potential response that ruthenium (II) produces, as blank.
Step a gained DNA interface and 10
-2-10
-9after M atriphos standard solution effect 1h, be then placed in 1mg/mL graphene solution 2h, interface is separated with solution again, is placed in 1mL10 with the reacted DNA of above-mentioned standard solution interface
-5mole dichloro three (1,10-phenanthrolene) in ruthenium (II) solution, act on 1 hour, interface is separated with solution, and working electrode is placed in to separated rear solution, dichloro three (1,10-phenanthrolene) ruthenium (II) interacts with Graphene, measure in solution and remain the potential response that dichloro three (1,10-phenanthrolene) ruthenium (II) produces, as the potential response of standard solution.
According to remaining potential response that dichloro three (1,10-phenanthrolene) ruthenium (II) produces in standard solution in the potential change signal Criterion working curve of blank difference.
(c) mensuration of the testing sample of unknown concentration atriphos:
Step a gained DNA interface is placed in 1mg/mL graphene solution 2h, is then placed in 1mL10 again
-5in mole dichloro three (1,10-phenanthrolene) ruthenium (II) solution, act on 1 hour, interface is separated with solution, and working electrode is placed in to separated rear solution, measures in solution and remains dichloro three (1,10-phenanthrolene) potential response that ruthenium (II) produces, as blank.
After the testing sample solution effect 1h of step a gained DNA interface and unknown concentration atriphos, and be placed on 2h in 1mg/mL graphene solution, interface is separated with solution, is placed in 1mL10 with the reacted DNA of above-mentioned testing sample interface
-5mole dichloro three (1,10-phenanthrolene) in ruthenium (II) solution, act on 1 hour, interface is separated with solution, and working electrode is placed in to separated rear solution, dichloro three (1,10-phenanthrolene) ruthenium (II) interacts with Graphene, measure in solution and remain the potential response that dichloro three (1,10-phenanthrolene) ruthenium (II) produces, as the potential response of testing sample.
In testing sample solution, remain the potential response of dichloro three (1,10-phenanthrolene) ruthenium (II) generation in blank difference, difference by standard working curve, finally obtains recording atriphos concentration in testing sample again.
Embodiment amplifying nucleic acid is fit also can be by heavy metal ion as potassium, mercury, lead, copper, zinc, uranium; Organic dyestuff is as sulfo group rhodamine B, reactive dark green KE-4BD; Amino acid is as arginine, TYR amine; Organic molecule is as ***e, cholic acid, Aspartame (containing phenylalanine), (R)-Thalidomide, 17 beta estradiols, bisphenol-A, buttocks worm amidine; Nucleic acid is as atriphos, adenylate, adenosine diphosphate (ADP); RNA is as TAR-RNA; Polysaccharide is as cellobiose, saliva lactose; Enzyme is as fibrin ferment, protein kinase, centriole cell elastase, HIV1-RT; Toxin is as ricin (WA); Growth factor is as basic fibroblast growth factor, platelet-derived growth factor-B chain; Antibody is as human immunoglobulin(HIg) IgE; All kinds of aptamers that bacterium and cell are part as spore, lung carcinoma cell, pathogenic bacteria are replaced.
Meanwhile, Graphene carrier can also adopt carbon nano-tube, molybdenum disulfide or tungsten disulfide to replace.
Embodiment 7
The working electrode that adopts embodiment 4, the detection for DNA, is specially:
Detection method is as follows:
A. the magnetic Nano of 50ul isosulfocyanate radical being modified is placed in DNA identification probe (this probe end part is complementary with DNA base sequence to be measured) solution, 37 degree reactions 1 hour, magnetic resolution, the solution that more above-mentioned aptamer modified magnetic Nano is placed in to 5% bovine serum albumin(BSA) reacts 1 hour, and magnetic resolution obtains DNA functional magnetic nano particle.DNA functional magnetic nano particle is reacted 2 hours with auxiliary DNA solution, on magnetic Nano surface, there is hybridization chain reaction, magnetic resolution, is placed in DNA hybridization buffer solution (10mM Tris-HCl+1mM EDTA+500mM NaCl+1mM MgCl by magnetic Nano
2, pH7.4) in, built aptamer functionalization double-stranded DNA interface.
Wherein, auxiliary DAN solution, for assisting DNA2 and DNA3 reaction after 2 hours, adds auxiliary DNA1 to obtain.
(b) Criterion working curve:
30 μ L step a gained double-stranded DNA interfaces, magnetic resolution is placed on 1mL10
-5in mole dichloro three (1,10-phenanthrolene) ruthenium (II) solution, act on 1 hour, interface is separated with solution, and working electrode is placed in to separated rear solution, measures in solution and remains dichloro three (1,10-phenanthrolene) potential response that ruthenium (II) produces, as blank.
Step a gained double-stranded DNA interface and 10
-6-10
-18after mole DNA effect 1h to be measured, interface is separated with solution, is placed in 1mL10 with the reacted double-stranded DNA of above-mentioned standard solution interface
-5mole dichloro three (1,10-phenanthrolene) in ruthenium (II) solution, act on 1 hour, interface is separated with solution, working electrode is placed in to separated rear solution, measure in solution and remain the potential response that dichloro three (1,10-phenanthrolene) ruthenium (II) produces, as the potential response of standard solution.
According to remaining potential response that dichloro three (1,10-phenanthrolene) ruthenium (II) produces in standard solution in the potential change signal Criterion working curve of blank difference.
(c) mensuration of the testing sample of unknown concentration DNA:
After 30 μ L step a gained double-stranded DNA INTERFACE MAGNETISM separation, be placed in 1mL10
-5in mole dichloro three (1,10-phenanthrolene) ruthenium (II) solution, act on 1 hour, interface is separated with solution, and working electrode is placed in to separated rear solution, measures in solution and remains dichloro three (1,10-phenanthrolene) potential response that ruthenium (II) produces, as blank.
After the separation of step a gained double-stranded DNA INTERFACE MAGNETISM with testing sample effect 1h after, interface is separated with solution, is placed in 1mL10 with the reacted double-stranded DNA of above-mentioned testing sample interface
-5mole dichloro three (1,10-phenanthrolene) in ruthenium (II) solution, act on 1 hour, interface is separated with solution, working electrode is placed in to separated rear solution, measure in solution and remain the potential response that dichloro three (1,10-phenanthrolene) ruthenium (II) produces, as the potential response of testing sample
In testing sample solution, remain the potential response of dichloro three (1,10-phenanthrolene) ruthenium (II) generation in blank difference, difference, again by standard working curve, finally obtains recording the concentration of DNA in testing sample.
In described step a, HCR reaction following (as shown in Figure 5):
(1) isosulfocyanate radical end magnetic particle solution is purchased from Xi'an gold magnetic nano biological science and technology limited Company (xMag
tM).In experiment, according to kit standard program, carry out aptamer mark.Get 2 1.5mL centrifuge tubes and be once labeled as 1,2, then add respectively the good xMag of 30 μ L mark
tMisosulfocyanate radical end magnetic particle solution, magnetic resolution is removed supernatant.
(2) in centrifuge tube 1, add 30 microlitre DNA hybridization buffer solution (10mM Tris-HCl+1mM EDTA+500mM NaCl+1mM MgCl
2, pH7.4),, in centrifuge tube 2, add 30 microlitre DNA1 liquid, in 37 ℃ of water-baths, react 1h, then magnetic resolution is got supernatant.
(3), for the centrifuge tube in step 2, add 40 microlitre DNA hybridization buffer solution (10mM Tris-HCl+1mM EDTA+500mM NaCl+1mM MgCl in 1
2, pH7.4), add 40 microlitre DNA2 and DNA3 hybridization reaction solution (adding front hybridization reaction time 2h) in 2, in 37 ℃ of water-baths, react 1h, then magnetic resolution is removed supernatant.Wherein bisphenol-A aptamer sequence is as follows: 5'-GGG CCG TTC GAA CAC GAG CAT GCC GGT GGG TGG TCA GGT GGG ATA GCG TTC CGC GTA TGG CCC AGC GCA TCA CGG GTT CGC ACC AGG ACA GTA CTC AGG TCA TCC TAG-3', the sequence of auxiliary DNA1: 3'-GCG TGG TCC TGT CAT GAG TCC AGT AGG ATC ACT AAA AGG GTC TGA GGG-5'; The sequence of auxiliary DNA2: 5'-TGA TTT TCC CAG ACT CCC CGT GGA CCC CCT CAT-3'; The sequence of auxiliary DNA3: 3'GCA CCT GGG GGA GTA ACT AA A AGG GTC TGA GGG-5'.
In the present embodiment step a, the sequence of DNA can design according to DNA sequence dna to be measured.
Embodiment 8
Detection method is as follows:
Current potential sensing: comprise working electrode 1, contrast electrode 2 and potential measurement instrument 3, potential measurement instrument 3 connects respectively working electrode 1 and outer contrast electrode 2 by wire, working electrode 1 and outer contrast electrode 2 insert and fill in the detection cell 4 that detects liquid, working electrode 1 is for being fixed with the solid electrode of polymer sensitive membrane 5, in detection cell 4, be provided with magnetic stirring 6 with beating action, supporting at the bottom of detection cell have magnetic stirring apparatus to rotate magnetic stir bar (referring to Fig. 2).
The polymer film of the cationite tetramethylphenyl sodium borate of take doping is working electrode sensitive membrane, and silver-silver chloride electrode is outer contrast electrode, selects " open circuit potential-time " technology to measure potential value.
By 10 μ L25mmol/LPOT-CH
2cl
2solution drips in copper electrode surface, after bone dry again by 50mg (0.5mg tetramethylphenyl sodium borate wherein, four (dodecyl)-tetra-(4-chlorphenyl) ammonium borates (ETH500) 5mg, polymer film methyl methacrylate-certain herbaceous plants with big flowers ylmethyl methyl acrylate 44.5mg) membrane component is dissolved in 0.5mL methylene chloride (in advance logical nitrogen deoxygenation), stirs after two hours, lead to again after nitrogen deoxygenation standby.Get the above-mentioned membrane material of 100 microlitre and drip on the solid copper electrode of polishing, and more than this electrode is placed in to thermostatic drying chamber volatile dry 12h.Dried electrode can be soaked in 10
-3moL/L Ru
2+in solution, activate 24h, 10
-9moL/L Ru
2+in solution, activate after 24h for measuring.
The present embodiment is for the detection of lgG, and testing process is as follows:
By 1mL xMag
tMisosulfocyanate radical end magnetic particle magnetic resolution is placed in 1mL goat-anti mouse lgG antibody-solutions, 37 degree reactions 0.5 hour, employing cleaning fluid cleans, magnetic resolution, the solution that more above-mentioned aptamer modified magnetic Nano is placed in to 5% bovine serum albumin(BSA) reacts 1 hour, and magnetic resolution obtains the magnetic nanoparticle of antibody labeling.
(b) Criterion working curve:
The magnetic nanoparticle of 20 μ L antibody labelings, after magnetic resolution, (500mL contains 4g NaCl, 0.1g KCl, 0.72g Na with 20 μ L immune response damping fluids
2hPO
4, 0.12gKH
2pO
4), immune response one hour, magnetic resolution adds the goat-anti mouse lgG antibody of 20 μ L DNA markers, immune response one hour.After magnetic resolution, add in the DNA with marker DNA complementation and react one hour, magnetic resolution is placed on 1mL10
-6in mole dichloro three (1,10-phenanthrolene) ruthenium (II) solution, act on 1 hour, interface is separated with solution, and working electrode is placed in to separated rear solution, measures in solution and remains dichloro three (1,10-phenanthrolene) potential response that ruthenium (II) produces, as blank.
The magnetic nanoparticle of 20 μ L antibody labelings, after magnetic resolution with 20 μ L10
-6-10
6(500mL contains 4g NaCl to ng/mL lgG standard solution, 0.1g KCl, 0.72gNa
2hPO
4, 0.12g KH
2pO
4), immune response one hour, magnetic resolution adds the goat-anti mouse lgG antibody of 20 μ L DNA markers, immune response one hour.After magnetic resolution, add in the DNA with marker DNA complementation and react one hour, magnetic resolution is placed on 1mL10
-5mole dichloro three (1,10-phenanthrolene) in ruthenium (II) solution, act on 1 hour, interface is separated with solution, working electrode is placed in to separated rear solution, measure in solution and remain the potential response that dichloro three (1,10-phenanthrolene) ruthenium (II) produces, as the potential response of standard solution.
According to remaining potential response that dichloro three (1,10-phenanthrolene) ruthenium (II) produces in standard solution in the potential change signal Criterion working curve of blank difference.
(c) mensuration of the testing sample of unknown concentration lgG:
The magnetic nanoparticle of 20 μ L antibody labelings, after magnetic resolution, (500mL contains 4g NaCl, 0.1g KCl, 0.72g Na with 20 μ L immune response damping fluids
2hPO
4, 0.12g KH
2pO
4), immune response one hour, magnetic resolution adds the goat-anti mouse lgG antibody of 20 μ L DNA markers, immune response one hour.After magnetic resolution, add in the DNA with marker DNA complementation and react one hour, magnetic resolution is placed on 1mL10
-6in mole dichloro three (1,10-phenanthrolene) ruthenium (II) solution, act on 1 hour, interface is separated with solution, and working electrode is placed in to separated rear solution, measures in solution and remains dichloro three (1,10-phenanthrolene) potential response that ruthenium (II) produces, as blank.
The magnetic nanoparticle of 20 μ L antibody labelings, after magnetic resolution with 20 μ L unknown sample solution, immune response one hour, magnetic resolution adds the goat-anti mouse lgG antibody of 20 μ L DNA markers, immune response one hour.After magnetic resolution, add in the DNA with marker DNA complementation and react one hour, magnetic resolution is placed on 1mL10
-5mole dichloro three (1,10-phenanthrolene) in ruthenium (II) solution, act on 1 hour, interface is separated with solution, working electrode is placed in to separated rear solution, measure in solution and remain the potential response that dichloro three (1,10-phenanthrolene) ruthenium (II) produces, as the potential response of testing sample.
In testing sample solution, remain the potential response of dichloro three (1,10-phenanthrolene) ruthenium (II) generation in blank difference, difference, again by standard working curve, finally obtains recording the concentration of lgG in testing sample.
In the present embodiment if cationite can be sodium tetraphenylborate (tetraphenylborate), tetramethylphenyl sodium borate (tetra (p-tolyl) borate), four ptolylboronic acid sodium, four (the fluorine-based phenyl of 4-) tetrakis sodium borate (4-fluorophenyl), 43,5-two (1,1,1, methoxyl-2,3,3,3-perfluoro-2 propyl group phenyl) sodium borate (tetrakis-[3,5-bis (1,1,1,3,3,3-hexafluoro-2-methoxy-2-prop yl) phenyl]-borate).
Antibody in the present embodiment can, according to the difference of test substance, select corresponding antibody and DNA marker anti-.
Claims (10)
1. one kind is detected dichloro three (1,10-phenanthrolene) potentiometric sensor of ruthenium (II), comprise working electrode, contrast electrode and potential measurement instrument, it is characterized in that: potential measurement instrument connects respectively working electrode and outer contrast electrode by wire, working electrode and outer contrast electrode insert and fill in the detection cell that detects liquid, working electrode bottom is provided with polymer sensitive membrane, is provided with the propulsion system with disturbance solution effects in detection cell;
Described polymer sensitive membrane is mixed by the ratio of weight and number of 20-40:40-80:0.1-10:0.1-10 by polymeric matrix material, plastifier and cationite, obtains resilient polymer sensitive membrane;
Described polymeric matrix material is Polyvinylchloride, carboxylated Polyvinylchloride, poly-butylacrylic acid ester, butyl polyacrylate, polyetherimide, rubber, sol-gel film, methyl methacrylate-certain herbaceous plants with big flowers ylmethyl methyl acrylate copolymer or n-butyl acrylate-hydroxyethyl methylacrylate multipolymer; Plastifier is o-nitrobenzene octyl ether, two-2-ethylhexyl ester in the last of the ten Heavenly stems, dibutyl sebacate or di-n-octyl sebacate; Cationite is four (3,5-bis-(trifluoromethyl) phenyl) sodium borate, dinonylnaphthalene sulfonic acid or dinonylnaphthalene sulfonic acid salt or borate.
2. by the potentiometric sensor of detection dichloro three claimed in claim 1 (1,10-phenanthrolene) ruthenium (II), it is characterized in that: between described working electrode and polymer sensitive membrane, be provided with conducting stratum material.
3. by the potentiometric sensor of detection dichloro three (1, the 10-phenanthrolene) ruthenium (II) described in claim 1 or 2, it is characterized in that: described working electrode is liquid electrode or solid electrode; Wherein, liquid working electrode fills internal-filling liquid and is inserted with the electrode of internal reference electrode in being.
4. by detection dichloro three (1 claimed in claim 3, 10-phenanthrolene) potentiometric sensor of ruthenium (II), it is characterized in that: described solid working electrode is carbon electrode, gold electrode, copper electrode, silver electrode, platinum electrode, indium-tin oxide electrode, zinc oxide electrode, silicon electrode, carbon fiber electrode, silicon carbide electrode, porous gold electrode, carbon nano tube modified electrode, gold nano modified electrode, platinum Nanoparticle Modified Electrode, graphene modified electrode, C60 modified electrode, porous carbon modified electrode, carbon screen printing electrode, gold screen printing electrode, refill plate electrode or ceramic chip electrode,
Described conducting stratum material is 3-alkylthrophene, polypyrrole, polythiophene, polyaniline, polyacetylene, poly-n-octyl thiophene, six polythiophenes, poly-ethylenedioxy thiophene, carbon nano-tube, Graphene, platinum nanometer, gold nano, molybdenum disulfide, mesoporous carbon, porous gold or tin indium oxide nanometer.
5. one kind is detected dichloro three (1,10-phenanthrolene) method of ruthenium (II), it is characterized in that: the working electrode of described potentiometric sensor and the insertion of outer contrast electrode are contained to dichloro three (1 to be measured, 10-phenanthrolene) in the detection liquid of ruthenium (II), by potential measurement instrument, measure the potential change on polymer sensitive membrane on working electrode, according to potential change signal and standard working curve, record dichloro three to be measured (1,10-phenanthrolene) ruthenium (II) concentration.
6. an application that detects the potentiometric sensor of dichloro three (1,10-phenanthrolene) ruthenium (II), is characterized in that: described potentiometric sensor can be used as the biology sensor based on DNA or aptamer the detection quantitative to different target molecules.
7. by detection dichloro three (1 claimed in claim 6, 10-phenanthrolene) application of the potentiometric sensor of ruthenium (II), it is characterized in that: using aptamer as identification molecule, dichloro three (1, 10-phenanthrolene) ruthenium (II) is as embedding molecule, aptamer and complementary DNA form double-stranded DNA, dichloro three (1, 10-phenanthrolene) ruthenium (II) embeds double-stranded DNA, by the potentiometer in described potentiometric sensor, measure the indication ion dichloro three (1 causing after target molecule and aptamer effect, 10-phenanthrolene) potential change that ruthenium (II) concentration change causes, according to potential change signal and standard working curve, record target molecule testing concentration.
8. by the application of the potentiometric sensor of detection dichloro three claimed in claim 7 (1,10-phenanthrolene) ruthenium (II), it is characterized in that: described aptamer is incorporated in liquid to be detected, or be incorporated in and detect in liquid after fixing with substrate.
9. by detection dichloro three (1 claimed in claim 8, 10-phenanthrolene) application of the potentiometric sensor of ruthenium (II), it is characterized in that: described fixed nucleic acid is fit and the substrate of complementary DNA is ferroferric oxide magnetic nanoparticle, at the bottom of magnetic graphite thiazolinyl, carbon substrate, gold substrate, copper substrate, at the bottom of money base, platinum substrate, tin indium oxide substrate, at the bottom of Zinc oxide-base, carbon fiber substrates, silicon carbide substrate, porous gold substrate, carbon nano tube modified substrate, gold nano is modified substrate, the nano-modified substrate of platinum, graphene modified substrate, C60 modifies substrate, porous carbon is modified the surfaces such as substrate, carbon filament reticulated printing electrode basement, spun gold reticulated printing electrode basement, the substrate of refill plate electrode or ceramic chip electrode basement.
10. by detection dichloro three (1 claimed in claim 8,10-phenanthrolene) application of the potentiometric sensor of ruthenium (II), is characterized in that: it is that aptamer and wall scroll complementary strand form double-stranded DNA that described aptamer and complementary DNA form double-stranded DNA; Or aptamer forms long double-stranded DNA by circulation isothermal strand displacement polyreaction and hybridization chain reaction (Hybridization chain reaction, HCR).
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| CN107402249A (en) * | 2016-05-21 | 2017-11-28 | 云南民族大学 | A kind of method based on hybridization chain reaction signal amplification technique detection metal ion |
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| CN109374710A (en) * | 2018-10-30 | 2019-02-22 | 浙江大学 | A kind of all solid state ion selective electrode and preparation method thereof based on conductive tungsten disulfide nano slices modified electrode |
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| CN116989652B (en) * | 2023-09-25 | 2023-12-08 | 科瑞工业自动化***（苏州）有限公司 | Displacement sensor capable of being used as reference electrode and using method thereof |
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